Crosslinking of Biological Material
Crosslinking of biological molecules is often desired for optimum effectiveness in biomedical applications. For example, collagen, which constitutes the structural framework of biological tissue, has been extensively used for manufacturing bioprostheses and other implanted structures, such as vascular grafts, wherein it provides a good medium for cell infiltration and proliferation. More recently, collagenous material, such as collagen, elastin or gelatin is used as drug carriers. However, biomaterials derived from collagenous tissue or collagenous material must be chemically modified and subsequently sterilized before they can be used in humans. The fixation, or crosslinking, of collagenous material increases biodurability and reduces antigenicity and immunogenicity. In one aspect of the present invention, crosslinking of a drug-containing biological material with genipin enables the resulting material (“biological substance”) with less antigenicity or immunogenicity, wherein the biological material comprises collagen, gelatin, elastin, chitosan, NOCC, and the like that has at least one amino functional group for reaction with genipin.
Clinically, biological tissue material has been used in manufacturing heart valve prostheses, small-diameter vascular grafts, biological patches, venous valve bioprostheses, bioadhesives, ligament replacement, stent coverings, and wound dressings, among others. However, the biological tissue material has to be fixed with a crosslinking or chemically modifying agent and subsequently sterilized before they can be used in humans. The fixation of biological tissue or collagen is to reduce antigenicity and immunogenicity and prevent enzymatic degradation. Various crosslinking agents have been used in fixing biological tissue. These crosslinking agents are mostly synthetic chemicals such as formaldehyde, glutaraldehyde, dialdehyde starch, glyceraldehydes, cyanamide, diimides, dimethyl adipimidate, diisocyanates, and epoxy compound. However, these chemicals are all highly cytotoxic which may impair the biocompatibility of biological tissue. Of these, glutaraldehyde is known to have allergenic properties, causing occupational dermatitis and is cytotoxic at concentrations greater than 10-25 ppm and as low as 3 ppm in tissue culture. It is therefore desirable to provide a crosslinking agent (synonymous to a crosslinking reagent) suitable for use in biomedical applications that is within acceptable cytotoxicity and that forms stable and biocompatible crosslinked products.
An example of a genipin-crosslinked heart valve is reported by Sung et al., a co-inventor of the present invention, (Journal of Thoracic and Cardiovascular Surgery vol. 122, pp. 1208-1218, 2001) entitled Reconstruction of the right ventricular outflow tract with a bovine jugular vein graft fixed with a naturally occurring crosslinking agent (genipin) in a canine model, entire contents of which are incorporated herein by reference. Sung et al. herein discloses genipin and its crosslinking ability to a collagen-containing biological tissue heart valve.
To achieve this goal, a naturally occurring crosslinking agent (genipin) has been used to fix biological tissue. The co-pending application Ser. No. 09/297,808 filed Sep. 27, 2001, entitled “Chemical modification of biomedical materials with genipin” is incorporated and cited herein by reference. The cytotoxicity of genipin was previously studied in vitro using 3T3 fibroblasts, indicating that genipin is substantially less cytotoxic than glutaraldehyde (Sung H W et al., J Biomater Sci Polymer Edn 1999; 10:63-78). Additionally, the genotoxicity of genipin was tested in vitro using Chinese hamster ovary (CHO-K1) cells, suggesting that genipin does not cause clastogenic response in CHO-K1 cells (Tsai C C et al., J Biomed Mater Res 2000; 52:58-65), incorporated herein by reference. A biological material (including collagen, elastin, chitosan, or gelatin-containing substrate) treated with genipin resulting in acceptable cytotoxicity is key to biomedical applications.
Sung and Liang in U.S. Pat. No. 6,545,042 entitled Acellular Biological Material Chemically Treated with Genipin, entire contents of which are incorporated herein by reference, disclose an acellular tissue providing a natural microenvironment for host cell migration to accelerate tissue regeneration. The genipin-treated biological biomaterial has reduced antigenicity and immunogenicity.
Kyogoku et al. in U.S. Pat. No. 5,037,664, U.S. Pat. No. 5,270,446, and EP 0366998 teach the crosslinking of amino group containing compounds with genipin and the crosslinking of genipin with chitosan. They also teach the crosslinking of iridoid compounds with proteins which can be vegetable, animal (collagen, gelatin) or microbial origin. However, they do not teach the use of the crosslinked products as biocompatible drug carriers.
Smith in U.S. Pat. No. 5,322,935 teaches the crosslinking of chitosan polymers and then further crosslinking again with covalent crosslinking agents like genipin and glutaraldehyde. Smith, however, does not teach the use of the cross-linked products as biocompatible drug carriers.
Drugs for Therapeutic Use
In an attempt to prevent restenosis or reduce intimal smooth muscle cell proliferation following angioplasty, numerous pharmaceutical agents have been employed clinically, concurrent with or following angioplasty. Most pharmaceutical agents employed in an attempt to prevent or reduce the extent of restenosis have been unsuccessful. The following list identifies several of the agents for which favorable clinical results have been reported: lovastatin (Sahni, R., Circulation 1989; 80 (Suppl.):65; Gellman, J., J. Am. Coll. Cardiol. 1991; 17:251); thromboxane A2 synthetase inhibitors such as DP-1904 (Yabe, Y., Circulation 1989; 80 (Suppl.):260); eicosapentanoic acid (Nye, E., Aust. N.Z. J. Med. 1990; 20:549); ciprostene (a prostacyclin analog) (Demke, D., Brit. J. Haematol 1990; 76 (Suppl.):20; Darius, H., Eur. Heart J. 1991; 12 (Suppl.):26); trapidil (a platelet derived growth factor) (Okamoto, S., Circulation 1990; 82 (Suppl.):428); angiotensin convening enzyme inhibitors (Gottlieb, N., J. Am. Coll. Cardiol. 1991; 17 (Suppl. A):81A); and low molecular weight heparin (de Vries, C., Eur. Heart J. 1991; 12 (Suppl.):386), entire contents of the above-referred drugs and their therapeutic effects are incorporated herein by reference. It is one aspect of the present invention to provide site-specific administration of the pharmaceutical agents disclosed in this invention to the target site for effective therapy via a genipin-crosslinked chitosan, collagen, elastin or gelatin-containing biological microspheres carrier.
Many compounds have been evaluated in a standard animal model. The immunosuppressive agent cyclosporin A has been evaluated and has produced conflicting results. Jonasson reported that cyclosporin A caused an inhibition of the intimal proliferative lesion following arterial balloon catheterization in vivo, but did not inhibit smooth muscle cell proliferation in vitro. (Jonasson, L., Proc. Natl. Acad. Sci. 1988; 85:2303). Ferns reported that when de-endothelialized rabbits were treated with cyclosporin A, no significant reduction of intimal proliferation was observed in vivo. Additionally, intimal accumulations of foamy macrophages, together with a number of vacuolated smooth muscle cells in the region adjacent to the internal elastic lamina were observed, indicating that cyclosporin A may modify and enhance lesions that form at the sites of arterial injury. (Ferns, G. A., Circulation 1989; 80 (Supp): 184; Ferns, G., Am. J. Path. 1990; 137:403).
Morris et al. in U.S. Pat. No. 5,516,781 disclosed Rapamycin (also known as sirolimus), a macrocyclic triene antibiotic produced by Streptomyces hygroscopicus that has been shown to prevent the formation of humoral (IgE-like) antibodies in response to an albumin allergic challenge (Martel, R., Can. J. Physiol. Pharm. 1977; 55:48), inhibit murine T-cell activation (Staruch, M., FASEB 1989; 3:3411), prolong survival time of organ gratis in histoincompatible rodents (Morris, R., Med. Sci. Res. 1989; 17:877), and inhibit transplantation rejection in mammals. Rapamycin blocks calcium-dependent, calcium-independent, cytokine-independent and constitutive T and B cell division at the G1-S interface. Rapamycin inhibits gamma-interferon production induced by I1-1 and also inhibits the gamma-interferon induced expression of membrane antigen. (Morris, R. E., Transplantation Rev. 1992; 6:39). The use of rapamycin in preventing coronary graft atherosclerosis (CGA) in rats has been disclosed by Meiser (J. Heart Lung Transplant 1990; 9:55). Arterial thickening following transplantation, known as CGA, is a limiting factor in graft survival that is caused by a chronic immunological response to the transplanted blood vessels by the transplant recipient's immune system (Dec. G, Transplantation Proc. 1991; 23:2095 and Dunn, M. Lancet 1992; 339:1566).
Further, Morris et al. in U.S. Pat. No. 5,516,781 claims a new use of rapamycin for preventing CGA, in that CGA does not involve injury to the recipients' own blood vessels; it is a rejection type response. The disclosed patent '781 is related to vascular injury to native blood vessels. The resulting intimal smooth muscle cell proliferation does not involve the immune system, but is growth factor mediated. For example, arterial intimal thickening after balloon catheter injury is believed to be caused by growth factor (PGDF, bFGF, TGFb, IL-1 and others)-induced smooth muscle cell proliferation and migration. (Ip, J. H., J. Am. Coll. Cardiol 1990; 15:1667). Ferns has also shown that the immune response is not involved in arterial intimal thickening following balloon catheterization, as he found that there was no difference in intimal thickening between arteries from athymic nude rats (rats lacking T-cells) and normal rats after balloon catheterization (Am. J. Pathol. 1991; 138:1045). The above-cited patent (U.S. Pat. No. 5,516,781) and literatures are incorporated herein by reference.
In the past, polymer or plastic materials have been used as a carrier for depositing a drug or pharmaceutical agent onto the periphery of a stent to treat restenosis. One example is U.S. Pat. No. 5,886,016 to Hunter et al., entire contents of which are incorporated herein by reference. Hunter et al. discloses a method for treating a tumor excision site, comprising administering to a patient a composition comprising paclitaxel, or an analogue or derivative thereof, to the resection margin of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at said site is inhibited. The composition further comprises a polymer, wherein the polymer may comprise poly (caprolactone), poly(lactic acid), poly(ethylene-vinyl acetate), and poly(lactic-co-glycolic) acid.
In another example, Biocompatibles PC (phosphorylcholine by Biocompatibles) has been added as a drug carrier or surface modifier for treating tissue injury due to angioplasty and/or stenting. The technique comprises a hydrophobic component that aids in the initial adhesion and film-formation of the polymer onto the stainless steel stent substrate, and other groups allow cross-linking both within the polymer and with the stent surface to achieve firm anchorage. The coating is thus tenaciously adhered to the stent and can survive balloon expansion without damage. A therapeutic drug can be loaded within the coated substrate, such as phosphorylcholine. In another aspect of the invention, PC can be loaded into a biological material (gelatin, elastin, collagen, or chitosan) and crosslinked with genipin as a microsphere drug carrier or onto a stent.
Drugs are usually admixed or entrapped physically within the polymer framework for slow drug release. The plastic polymer which is suitable as a drug carrier may not be biocompatible, whereas some biocompatible plastic polymer may not be able to contain a specific drug and release drug in an effective timely manner for effective therapies. Therefore, there is a clinical need to have a biocompatible drug carrier that releases an effective quantity of drug over a period of time for prolonged therapeutic effects.
In a co-pending patent application by two of the present co-inventors (Sung and Tu), Ser. No. 10/211,656 filed Aug. 2, 2002 entitled “Solidifiable Biological Material Chemically Treated with Genipin”, entire contents of which are incorporated herein by reference, discloses a collagen-drug-genipin compound coated onto a stent for treating proliferation and restenosis problems. In addition to collagen, the biological material or carrier may include gelatin, elastin, chitosan and the like.
In accordance with the present invention there is provided genipin treated gelatin microspheres loaded with drug for implant and other surgical applications which have shown to exhibit many of the desired characteristics important for optimal therapeutic function. In particular, the crosslinked gelatin-drug compound with drug slow release capability may be suitable as anti restenosis agent in treating atherosclerosis and other therapeutic applications.